Fig 1.
Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs).
(A) Results of the immunocytochemical analysis and the method of tissue printing. (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing, negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V. faba roots by the method of tissue printing, negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [pv; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns, mean from three independent experiments; bars, SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [pv; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns, mean from three independent experiments; bars, SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).
Fig 2.
Single strand breaks (SSBs) and double strand breaks (DSBs) were assessed with alkaline (pH ≥ 13) and neutral (pH = 10) variants of comet assay, respectively.
SSBs were generated during replication stress (i.e. after 2.5 mM hydroxyurea [HU] treatment) and DSBs were connected with the induction of premature chromosome condensation (PCC) due to co-treatment with HU and caffeine (CF). (A-F) fluorescence images of nuclei from individual protoplast of Vicia faba, stained with YOYO-1 after electrophoresis (comet assay); h, comet head; t, comet tail. (A-C) alkaline variant of comet assay dedicated to detection of SSBs; (D-F) neutral variant of comet assay assigned to DSBs. Scale bar in 'A' = 5 μm is applied to all figures presented. (A'-F') the intensity of DNA fluorescence after staining with YOYO-1 of the nuclei shown in (A-F). (A, D) nuclei of a protoplasts isolated from the untreated (control) V. faba roots. (B, E) nuclei of protoplasts isolated from the roots of V. faba treated with HU for 32 h. (C, F) nuclei of protoplasts isolated from the roots of V. faba treated with HU for 24 h and next co-treated with HU/CF for 8 h.
Fig 3.
Terminal deoxynucleotidyl-dUTP nick end labeling (TUNEL) assay in Click-iT technology in the untreated control (negative), DNase-treated control (positive), HU-treated and HU/CF-co-treated (i.e. PCC-induced) Vicia faba root meristem cells.
Left panel—DNA fragmentation in V. faba cells detected by TUNEL reaction and visualized by AlexaFluor 594. Central panel—DAPI stained nuclei. Right panel—Merged images (AlexaFluor 594 + DAPI). Positively stained nuclei appear in the DNase-treated cells (e.g. A-A'') and in the HU/CF co-treated cells (e.g. B-B''). Positively stained nuclei in the HU-treated series are indicated by arrowheads. Non-reacting nuclei can be seen in the negative control sections (as indicated in the highest panel described as 'CTRL'). Scale bar = 20 μm.
Fig 4.
Double in vivo staining with acridine orange (AO) and ethidium bromide (EB) as a useful tool for detecting and quantifying the state of dead, dying and living cells in root meristems of Vicia faba.
(A-A'') control. (B-B'') hydroxyurea-induced replication stress. (C-C'') caffeine-induced premature chromosome condensation (PCC). (A,B,C) fluorescence micrographs of nuclei in living (green), dying (range: yellow-to-orange), and dead (red) cells. (A',B',C') diagrams presenting the color spectrum resulting from measurements of the fluorescence intensity of nuclear chromatin stained with AO/EB, in order to determine the degree of damage in the nuclei of stressed roots of V. faba. (A'',B'',C'') circle diagrams presenting the percentage of living (green), dying (range: yellow-to-orange) and dead cells (red). The data shown in the pie charts in A'',B'',C'' indicate that the correlations were significant with reference to the number of dead cells for all experimental series reported herein: an association was found between the control and HU (p ≤ 0.05, Mann-Whitney U test), between the control and PCC (p ≤ 0.01, Mann-Whitney U test), and between the HU-treated and PCC-induced cells (i.e. HU/CF co-treated; p ≤ 0.01, Mann-Whitney U test). Scale bar in (A) = 20 μm is also applied to the figures presented in the pictures (B) and (C).
Fig 5.
Acridine orange (AO) and ethidium bromide (EB) staining of living, dying and dead cells in Vicia faba root meristem cells in relation to (A) the localization in particular zones, and (B-D) the surface area occupied by the cells in particular zones.
(A) Fluorescence picture of AO/EB stained V. faba roots in planta. (a) schematic figure presenting the outline of the roots of the control series, with marked (I) root cap, (II) zone of cell division i.e. root meristem, (III) zone of elongation, and the quiescent center. (b) the control roots, (c) the roots treated with 2.5 mM hydroxyurea (HU) for 32 h, (d) the roots treated with 2.5 mM HU for 24 h and then co-treated with 2.5 mM HU and 5 mM caffeine (CF). Scale bar = 1 mm. The schematic outline of the root from scheme (a) was placed over a root from the control series (b) on which the root outline from figure 'a' and figure 'b' are precisely overlapped, (c) a root from the series, in which seedlings were subjected to replication stress and (c) a root that was induced to premature chromosome condensation (PCC). (c-d) The continuous line marks those root fragments that in terms of size and shape were the same as the analogous areas in the roots of the control series (a-b versus c-d), while the broken line (in figures [c] and [d]) marks the root areas that indicated the appearance of aerenchymatic-like spaces that had formed in the roots that had been subjected to treatment with HU (c) or co-treated with the mixture of HU/CF (d). In places indicated by broken lines, roots of the series (c) and (d) were distinctly wider than the control (b). (B-D) quantitative presentation of surface area (%) occupied by the green, yellow-orange and red colors (that correspond to the populations of living, dying and dead cells, respectively) in the particular zones of V. faba roots. (B) quiescent center, (C) zone of cell division, i.e. root meristem, marked also as zone II, and (D) zone of elongation, marked as zone III.
Fig 6.
Electron micrographs of Vicia faba root meristem cells.
(A) control (32-h incubation in water); (B) hydroxyurea-treated (32-h); (C-C', D-D', E-F) hydroxyurea (HU) synchronized for 24 h and then HU/CF co-treated (for a successive 8 h; total incubation time: 32 h). The arrows in picture (C') point out vesicles of the Golgi apparatus. The arrows in picture (D') indicate lytic vacuoles localized in the vicinity of plasmodesmata. The square in the bottom right corner of picture (E) contains an enlarged image of the cell from picture (F). Asterisk (*), the visible light in the vacuoles presented in pictures (C and D) indicates the places of accumulation of deposits in the vacuoles, showing that these vacuoles function as lytic vacuoles. All the photos presented in figures (C-F) are derived from the series in which PCC was induced. However figure (C) shows the morphology of the root cuticle cells, from which the plastids seen in the picture (precisely amyloplasts, marked as 'p') are filled with statolith starch grains (marked as 's'). Successive figure (D) presents the appearance of a typical V. faba meristematic cell, whose morphology (apart from the deposits seen in the lytic vacuoles and indicated by the asterisk) does not significantly differ from the morphology of the control cells (comp. A and D). Two further pictures (E and F) illustrate the morphology of meristematic cells that entered the path of apoptosis-like programmed cell death (AL-PCD), while picture (E) shows premature vacuolization stadium, and picture (F) demonstrates: (1) extensive vacuolization within the whole meristematic cell space, (2) the presence of swollen ER compartments (indicated by arrows), and (3) the existence of autophagosome-like structures, created from ER (the structures inside the squares). a-l autophasome-like structure, c cytoplasm, cw cell wall, dch dense chromatin, ER endoplasmic reticulum, G Golgi structure, lv lytic vacuole, m mitochondrion, n nucleus, ne nuclear envelope, no nucleolus, nov nucleolus vacuole, p plastid, pd plasmodesmata, s starch, v vacuole. Scale bar = 5 μm.
Fig 7.
Spreading of (presumably) lytic enzymes from the interior of almost totally degenerated dead cells to adjacent cells through plasmodesmata.
(A-B) The direction of enzyme propagation is indicated with arrows. Double arrow-heads indicate cytoplasm residues pushed towards plasmalemma with suspended organelles already partially digested. The asterisk in picture (A) indicates electron lucent areas, newly formed inside a regular meristem cell (i.e. a cell that does not show changes in other regions, other than those marked with an asterisk where the wave of lytic enzymes arrived). Picture (B) Gradual, sequential propagation of a cleared up region appearing, presumably, due to the action of lytic enzymes in a cell neighboring a dead cell due to AL-PCD, towards further areas of the adjacent cell. c cytoplasm, cw cell wall, dch dense chromatin, lv lytic vacuole, m mitochondrion, mls multilamellar structure; n nucleus, p plastid, pd plasmodesmata, s starch; p(s) swollen plastids; m(s) swollen mitochondium. Scale bar = 5 μm.
Fig 8.
Scheme depicting experimental procedures used to reveal and to support the fact that the activation of vacuolar/autolytic type of plant-specific, apoptosis-like programmed cell death [(V/A) AL-PCD] is a secondary result of the caffeine-induced premature chromosome condensation (PCC) in hydroxyurea (HU) synchronized Vicia faba root meristem cells.
The scheme shows that PCC leads to the formation of cells with different phenotypes (phenotype A–typical of cells not diverging from the morphology of regular cells; phenotype B–characteristic of cells with the morphology typical of PCC induction from G2 phase; and phenotype C–cells with strongly fragmented chromosomes, induced to PCC, probably in early or middle sub-periods of S phase). The scheme shows that the induction of PCC is associated with the generation of DNA damage (mainly single-strain breaks [SSB] but also to a lesser extent, double-strain breaks [DSBs]). The occurrence of various types of DNA damage during PCC induction has been proven following the results of many experimental procedures (immunocytochemistry, with the use of antibodies against specific marker proteins, tissue printing methods, Western blot and comet tests–both alkaline and neutral). In turn, double staining with acridine orange and ethidium bromide allowed us to distinguish in planta, the sub-fractions of living, dying and dead cells. Preliminary histochemical analyses (classic staining with Schiff’s reagent in Feulgen’s method) showed the presence of such changes in the structure of cellular nuclei that could indicate the occurrence of apoptosis-like programmed cell death (AL-PCD). On the other hand, more detailed analyses, i.e. DNA electrophoresis and TUNEL reaction, confirmed by ultrastructural tests (performed at a level of transmission electron microscopy) proved the existence of vacuolar/autolytic type of AL-PCD (V/A-type AL-PCD) in stressed V. faba roots.